Project Summary/Abstract Over 2,500 children undergo hematopoietic cell transplantation (HCT) in the United States each year for an increasingly broad set of malignant and non-malignant diseases. Unfortunately, pulmonary complications after HCT are a leading cause of transplant-related mortality (TRM) and are frequently associated with a complex overlap of respiratory infection, viral reactivation, alloreactive inflammation, treatment-related lung injury, fluid overload, and other toxicities. In clinical practice, differentiation of pulmonary infection and non-infectious inflammation is challenging and failure to accurately diagnose pulmonary complications is associated with significant mortality. Further, an incomplete understanding of the varied biology of post-HCT pulmonary complications has to date limited the application of targeted therapeutics necessary to improve survival. Therefore, the goal of this K23 proposal is to advance our understanding of the pathobiology of post-HCT pulmonary complications in children. To do this, we will apply metagenomic sequencing technology to lower respiratory biospecimens from two geographically distinct pediatric cohorts. In Specific Aim 1a, we will determine the composition of the pulmonary microbiome in pediatric HCT patients with different subtypes of lung disease and will identify occult pathogens that can be implicated in disease. In Specific Aim 1b, we will model the outcome of transplant-related mortality (TRM) according to microbiomic characteristics while controlling for potentially confounding clinical traits. In Specific Aim 2a, we will analyze patterns of pulmonary gene expression in order to improve our understanding of dysregulated biological pathways involved in lung injury after HCT. In Specific Aim 2b, we will associate TRM with human gene expression signatures in order to identify high-risk patients with shared biology that may benefit from molecularly targeted interventions. In sum, these aims will develop a platform with which to more accurately diagnose pulmonary infections and identify discrete cellular pathways involved in infectious and non-infectious post-HCT lung injury. The knowledge gained from this investigation will be directly useful in improving diagnostic and prognostic classification schema necessary to adapt targeted therapeutics and improve patient outcomes. The candidate's career goal is to improve molecular characterization and treatment of pathobiologic subtypes of lung injury in children who have undergone HCT. In this K23 application, the candidate has proposed a detailed career development plan focused on developing a technical, methodological, and bioinformatics expertise in the execution of impactful research in this rapidly evolving cross-disciplinary field. The candidate is trained in Pediatric Critical Care Medicine, holds a faculty position at the University of California, San Francisco, and is well supported by an experience mentoring and scientific advising team. This K23 proposal will generate robust data to inform a subsequent R01 submission facilitating the candidate's transition to an independent scientific career aimed at improving outcomes for pediatric HCT patients with lung injury.